Biomarkers of endothelial dysfunction in treated and untreated rats with collagen induced rheumatoid arthritis
Background. High-grade chronic inflammation increases the risk for cardiovascular disease (CVD). Endothelial dysfunction is one of the earliest markers of inflammation-induced CVD. Treatment with anti-inflammatory drugs has shown improvement in endothelial function. However, the mechanisms whereby inflammation and anti-inflammatory drugs impact endothelial function are not well understood. Alterations in microRNA (miRNA), short non-coding RNAs that post-transcriptionally regulate gene expression, have been linked to various disease states. Evidence supports miRNAs as novel biomarkers and predictors of endothelial dysfunction and CVD. However, the effect of inflammation on miRNA expression and the mechanisms by which biologic anti-inflammatory treatments alter miRNA gene regulation to affect downstream signalling and ultimately endothelial function and CVD risk requires investigation.Methods. Three-month-old, male and female Sprague-Dawley rats were randomly divided into four groups. One group served as the control group (n=21) and received a saline subcutaneous injection. The other three groups were exposed to an arthritis inducing protocol where rats received a subcutaneous injection of bovine type-II collagen emulsified in incomplete Freund’s adjuvant at the base of the tail. Upon signs of inflammation, one group received no treatment (collagen induced arthritis (CIA) group, n= 27), one group received a tumour necrosis factor alpha (TNF-α) receptor blocker (anti-TNF-α group, n=13) every three days for six weeks and one group received an interleukin 6 (IL-6) receptor blocker (anti-IL-6 group, n=15) once a week for 6 weeks. Body weight, blood pressure and arthritis scores in the hind paws were measured throughout the study. At termination, vascular function was measured using applanation tonometry and vascular responses to vasodilators and vasoconstrictors in mesenteric and renal arteries (vascular reactivity) were measured using a wire myograph. Blood was obtained and serum concentrations of TNF-α, IL-6 and C-reactive protein (CRP) were measured by ELISA. Total RNA and total miRNA were extracted from cardiac tissue and serum, respectively, and reverse transcribed. Real time PCR was used to determine the relative expression of miR146a-5p, miR155-5p, vascular adhesion molecule-1 (VCAM-1) and pentraxin-3 (PTX-3). Group differences in body weight, blood pressure and arthritisscores were measured by repeated measures analysis of variance (ANOVA). Group differences in inflammatory cytokine concentrations were measured by a two-way ANOVA with a Tukey post-hoc test. Group differences in relative miRNA expression were measured by a Kruskal-Wallis test. Associations between relative miRNA expression and inflammatory markers, vascular function markers and cardiac function markers were determined by Pearson’s correlations. Results. Body weight and blood pressure did not change over the course of the study and did not differ between the groups (all p>0.05). Circulating inflammatory marker concentrations and arthritis scores were higher in all rats exposed to the collagen induced inflammatory protocol compared the control group (all p<0.05). The expression of VCAM-1 and PTX-3 were higher in the CIA and anti-IL-6 groups (both p<0.05), but not the anti- TNF-α group (p>0.05) compared to the control group. Relative miR146a-5p expression was higher in the CIA group compared to the anti-TNF-α group (p=0.01). Relative miR155-5p expression was higher in the CIA group compared to the control group (p=0.05). There were no differences in the relative expression of miR146a-5p or miR155-5p between the control and anti-TNF-α groups (p>0.05). Higher circulating CRP concentrations were associated with the relative expression of miR146a-5p (r=0.31, p=0.04) and miR155-5p (r=0.62, p=0.009), but the association was only seen in the absence of anti-inflammatory treatments. Relative miR146a-5p expression was associated with the sensitivity of mesenteric arteries to vasodilators in control and inflammation groups (r=0.39, p=0.04), but not when the anti-inflammatory treatments groups were included in the analysis (r=-0.09, p=0.52). Relative miR155-5p expressionwas associated with markers of arterial stiffness, renal artery sensitivity to vasodilators and maximal relaxation responses in the control and inflammation groups (all p<0.05), but not when treatment groups were included. Both miR146a-5p (r=0.35, p=0.008) and miR155-5p (r=0.31, p=0.02) were associated with relative mRNA expression of PTX-3, independent of anti-inflammatory treatment. Conclusion. Exposure to high-grade inflammation and TNF-α blocker treatment impact the expression of miR146a-5p and miR155-5p. Although miR155-5p was associated with markers of vascular reactivity and large artery function, these relationships are affected by anti-inflammatory treatment. The association between miR146a-5p and miR155-5p and endothelial function in a high-grade inflammatory model is not affected by anti-inflammatory treatment. Taken together, our results suggest that exposure to high-grade inflammation alters upstream gene regulation of endothelial function, which may be affected by TNF-α blocker treatment.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Psychology, University of the Witwatersrand, Johannesburg, 2020